Http://pinegenome.org/pinerefseq/ Fosmid-pools and 40-kb mate-pairs Children’s Hospital Oakland...

http://pinegenome.org/pinerefseq/

Fosmid-pools and 40-kb mate-pairs

Children’s Hospital Oakland Research Institute (CHORI)BACPAC Resources Center


Strategies for Sequencing large genomes with high-repeat content

A. Scaffolding of contigs (produced by WGS or Pool Seq strategy) with an abundant source of long-range (~40 kb) mate-pairs.

B. Reduced complexity: NextGen sequencing from E.coli clone pools with combined inserts sizes far below genome size. Creates contigs limited to the size of clones.


Vector to create 40-kbp mate pair libraries (“diTags”)

• Cloning of sheared genomic DNA in blunt-ended SnaBI site

• Includes options for creating mate-pairs by 5 strategies:

• Nicking, Nick translation, S1-nuclease (Nb.BbvCI or Nt.BspQI nicking sites)

• Fragment insert DNA with FspBI, Csp6I or both


Fragmenting fosmid inserts: nicking enzyme approach

• Nicking of vector (and insert)

• Nick-translate from vector into insert

• Convert nick into break

• Isolate vector plus attached insert-ends

• Ligate• PCT amplify• Illumina Sequence


Fragmenting fosmid inserts: use of 4-cutters

• Digest with 4-cutter

• Isolate vector with attached insert-ends

• Ligate• PCR-Amplify• Illumina

Sequence


Fragmenting fosmid inserts: use of FspBI, Csp6I or together


Reduced Complexity from E.coli clone pools

• Used by others to improve assemblies• Used also to derive long-haplotype

information• Ideally: larger clones (BACs) are more

desirable, more likely to span long “duplicons”• Economically: much cheaper to create large

fosmid libraries using sheared DNA (more random) with high genomic representation


Fosmid Pools• DNA extraction• Shearing (to average ~40 kbp)• Size-purification (pulsed-field gel

electrophoresis)• Ligation to excess vector

(dephosphorylated ends)• Packaging (extracts from mcrA, B, C

strains)• Determine titer of the “particle

library”• Create E.coli colonies (e.g. 1,000/150

mm Petri dish)• Isolate DNA from colonies and create

Illumina seq libraries• Quality Assessment: Quantitation &

Q-PCR (TAQMAN assays) to determine E.coli & fosmid vector contamination

Fosmid colonies: Documented & counted prior to pooling

Fosmid pools: DNA digested (PI-SceI); Lane 6: Undigested pool


DNA Isolation from fosmid Pools

• Midi-prep columns: high quality/quantity/cost• Small prep’s followed by amplification (Rolling Circle

Amplification, RCA)• Crude bacterial lysates purified using biotin-labeled

Triple-Helix probes (magnetic beads)• Triple-helix followed RCA

• Scalable? • Representative? • Artifact levels (e.g. chimeric reads? )• Costs?

Criteria:

Options:


Triple_Helix Purification of fosmid inserts*

• Removal of vector & E.coli contamination

• Scalable• Simple Purification

* Rapid isolation of cosmid insert DNA by triple-helix-mediated affinity capture. Ji H, Smith LM, Guilfoyle RA. Genet Anal Tech Appl. 1994;11(2):43-7. * Rapid restriction mapping of cosmids by sequence-specific triple-helix-mediated affinity capture. Ji H, Francisco T, Smith LM, Guilfoyle RA. Genomics. 1996 Jan 15;31(2):185-92.


Results• Newest vector now

in production: pFOSTH1.0

• Pools generated from midi-prep DNA, 12 pools sequenced

• Pools generated from mini-prep & RCA (enriched for E.coli mobile element: IS150)

• Pools now being generated with pFOSTH1.0

Fosmid pools in pFOSTH1.0 vector : PI-SceI -digested ; Lane 6: no digest


Acknowledgments

• Work at CHORI by Ann Holtz-Morris, Maxim Koriabine, Ph.D., and Boudewijn ten Hallers

• Loblolly DNA provided by Dana Nelson, Southern Institute of Forest Genetics

• Many collaborators within Loblolly Pine consortium

• PI: David Neale, UC Davis

(l to r) Co-PD Pieter de Jong, Ann Holtz-Morris, Maxim Koriabine, Boudewijn ten

Hallers

Http://pinegenome.org/pinerefseq/ Fosmid-pools and 40-kb mate-pairs Children’s Hospital Oakland...

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